In the field of cell analysis, cells are commonly placed in a multiwell microplate for purposes of testing multiple conditions and replicates in a single experiment. Standard microplates, such as 24- and 96-well plates, are two-dimensional arrays of wells. Such arrays include some wells that are at the border or edge of the array, i.e., in the first row, first column, last row, or last column. Border wells and non-border wells can experience different conditions; this is commonly known as an “edge effect”. Because such assays are typically conducted at mammalian body temperature (37° C.), and border wells are more exposed to the external environment, the environment within the border wells may be substantially different from that of the non-border wells. The evaporation of liquid from wells adjacent to the border of the plate occurs at a higher rate than that of non-border wells. This causes a temperature drop in the border wells due to evaporative cooling, resulting in an increase in the concentration of solutes in the liquid. Both the temperature differences and the concentration difference contribute to data inconsistency in these types of assays. Live-cell assays are particularly sensitive to these effects due to the dynamic nature of the assay and the sensitivity of living, metabolically active cells to the environmental conditions in which they are being measured. Examples of these types of assays include FLIPR calcium flux assays, Corning EPIC label-free assays, and certain high-content imaging assays.
Several solutions have been proposed and applied to such standard microplates to address this problem. One workaround is to sacrifice the use of the border wells in the assay. By simply filling them with fluid to the same height as the assay wells, the border wells provide a humidity buffer. This approach has serious drawbacks in that the capacity of the microplate is significantly diminished, and in the case of a 24-well plate more than half of the wells are sacrificed. As the size of the well array in the microplate decreases, a higher fraction of wells become border wells. At the extreme, in one-dimensional arrays, every well has a high rate of evaporation.
Another workaround is to seal the wells or plate by overlaying the assay wells with oil or wrapping the covered plate with a plastic paraffin film, such as Parafilm M® film available from Bemis Company, Inc., or similar material. One of the drawbacks to these methods is that gas exchange is reduced. Metabolically active cells require oxygen; thus restricting the supply of oxygen can be detrimental to the cells and cause changes in assay results.
Existing solutions to this problem include modifications to the instrumentation or the cell growth vessel, i.e., microplate and cover. A few instrumentation manufacturers attempt to mitigate these effects by putting humidity control into the measuring chambers in which the microplate is placed. In general, however, these options are rare as high humidity levels can cause problems with the instrument electronics.
Modifications to the cell growth vessel may include changes to the design of the microplate and lid. Changes to the lid include adding a moisture-holding layer to the lid. However, in the case of live-cell assays where addition of reagent during the course of the assay is required, a lid or cover cannot be used.
The addition of perimeter or border wells to the microplate provides an environmental buffer between the assay well and the ambient laboratory conditions. For example, a plate may have large edge troughs, e.g., four troughs, surrounding the array of wells. Fluid may be placed in each trough, thus providing an environmental buffer. A potential drawback of this design is the large volume of each trough. Because well plates are shallow, there is potential sloshing of the border fluid when the plate is tilted or moved around the laboratory. In addition, the depth of the troughs, being the same depth as that of the wells, may require that a significant amount of fluid, more than 10× the volume of the assay well, be added to each trough. Therefore the operator may need to use a different tool (such as a different volume pipet) to fill the border troughs and the assay wells.
Standard microplate designs include a lid or cover where the edge or skirt of the cover can be up to half the height of the plate itself and protrudes 1-2 millimeters (“mm”) beyond the wall of the plate. This may present a problem while handling these plates, as it takes some dexterity to consistently pick up both the plate and the lid off of a surface, e.g., to avoid accidentally picking up only the lid and thus exposing the contents of the plate. When dealing with cell cultures that must be maintained under sterile conditions, current plate and cover assembly designs introduce considerable risk to the integrity of the cultures. Similar risks apply to assays where the contents of the wells must be protected from ambient light.
Standard microplate designs have a fixed height and footprint, such that the volume of the wells varies with the number of wells arrayed in the plate. For example, a standard 384-well plate has four times as many wells as a standard 96-well plate, but each well is approximately one-fourth the volume. Likewise, as well density (i.e., wells per plate) goes down, the volume per well increases. This design, although convenient for maintaining a standard footprint, requires that the researcher use more cells and reagents per well when using a lower-density plate. In addition, the spacing between wells changes, which can be an inconvenience when adding reagents to the assay plate.
Presently, no microplate is commercially available for performing an assay on a fewer number of wells while maintaining standard volumes and well-to-well spacing. Maintaining these features and reducing the number of wells may require reducing the footprint. However, since many standard laboratory workflows and instruments are designed to this standard, an adapter or carrier of some sort would be required. Examples of instruments that accept standard-footprint microplates include plate readers, high content imaging systems, centrifuges, and automated plate handling robots.
Microscope slides adhere to a different standard in the lab, and some products exist that bridge the microplate and slide formats. Some commercially available slides contain assay wells fused to a glass microscope slide, providing assay wells with glass bottoms designed for high-resolution imaging on microscopes. Although they do provide wells, the dimensions of the wells vary and are not standard with respect to well-to-well spacing nor length and width dimensions.
A commercially available carrier for microscope slides that conforms to the Society for Laboratory Automation and Screening (“SLAS”) microplate footprint and height standards is designed for imaging applications, but the placement of the slides in the carrier allows for some variability in well position, which may make automated analysis challenging.